Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Epstein-Barr virus in gastric carcinomas with lymphoid stroma

AIMS: To clarify the relationship between the Epstein-Barr virus (EBV) and gastric carcinoma with lymphoid stroma (GCLS) in Koreans, and to characterize the EBV-positive GCLS. METHODS AND RESULTS: EBV infection was examined using EBER in-situ hybridization and polymerase chain reaction in 45 cases of GCLS among Koreans, and in 292 consecutive cases of gastric carcinomas without lymphoid stroma (non-GCLS) as controls. EBV infection was found in 30 tumours (67%) of GCLS and 10 tumours (3.4%) of non-GCLS (P < 0.05). EBV-positive GCLS was more prevalent in males, poorly differentiated histological type and diffuse type in Lauren's classification, and tended to be located more in the middle third of the stomach than EBV-negative GCLS (P < 0.05). p53 overexpression was observed in 22% of GCLS (17% of EBV-positive GCLS and 33% of EBV-negative GCLS), and 34% of non-GCLS (EBV-positive GCLS vs. non-GCLS: P = 0.056). The survival of the patient with GCLS was not correlated with EBV infection or p53 immunoexpression (follow-up period: 11-97 months). CONCLUSIONS: GCLS in Koreans is strongly associated with EBV infection. The prognosis in GCLS is not dependent upon either the status of EBV infection or the status of p53 immunoexpression.     

β-Adrenergic receptor coupled-adenylate cyclase of human fat cell ghosts

Summary The effects of beta-adrenergic agonists such as isoproterenol, norepinephrine and epinephrine upon the adenylate cyclase activity of human fat cell ghosts were tested, each alone and in combination with the beta-blocking agent propranolol. Saturating concentrations of these agents showed a 2–6.5-fold increase of enzyme activity without addition of any artificial cofactors. Isoproterenol was more potent in stimulating the enzyme system than epinephrine and nor-epinephrine. Propranolol caused a dose-dependent rightward shift of the log-dose response curve of these beta-adrenergic agonists. The assay of human fat cell adenylate cyclase in vitro may provide a simple and convenient assay system for the screening of beta-adrenergic drugs of potential therapeutic importance.Herrn Prof. Dr. Dr. h.c. G. Schettler zum 60. Geburtstag gewidmet     

A girl with 1p36 deletion syndrome and congenital fiber type disproportion myopathy

Chromosome 1p36 deletion syndrome is characterized by hypotonia, moderate to severe developmental and growth retardation, and characteristic craniofacial dysmorphism. Muscle hypotonia and delayed motor development are almost constant features of the syndrome. We report a 4-year-old Japanese girl with 1p36 deletion syndrome whose muscle pathology showed congenital fiber type disproportion (CFTD) myopathy. This is the first case report of 1p36 deletion associated with CFTD. This association may indicate that one of the CFTD loci is located at 1p36. Ski proto-oncogene −/− mice have phenotypes that resemble some of the features observed in patients with 1p36 deletion syndrome. Because fluorescent in situ hybridization analysis revealed that the human SKI gene is deleted in our patient, some genes in 1p36, including SKI proto-oncogene, may be involved in muscle hypotonia and delayed motor development in this syndrome. Received: March 4, 2002 / Accepted: July 7, 2002     

Activation and internalization of p56lck upon CD45 triggering of Jurkat cells

Relationships between CD45 and p56^Ick have been suggested by co-immunoprecipitation of both proteins and by dephosphorylation of the p56^lck regulatory site, Tyr 505, by CD45 in vitro. We investigated whether the kinase activity of p56^lck is modulated in T cells triggered via CD45. We showed that incubation of Jurkat cells with a combination of two anti-CD45 monoclonal antibodies (mAb) (MC5/2 + D3/9) induced an increase in p56^lck kinase activity, while a single mAb did not. Under these conditions, p56^lck underwent two consecutive waves of activation. This was accompanied by internalization of the kinase and by a time-dependent increased accessibility of CD45 phosphatase at the plasma membrane. Similarly, activation and internalization of p56^lck were observed using a combination of anti-CD45 (MC5/2) and anti-CD2(T11₂) mAb, suggesting that a functional complex consisting of CD45, CD2 and p56^lck was formed upon cell triggering. Taken together, these results suggests that: (i) CD45 participates in the regulation of p56^lck kinase activity in vivo and that (ii) CD45 could play a mediator role in the stimulation and endocytosis of p56^lck through the CD2 pathway.     

Adjustment of metabolism,catecholamines and β-adrenoceptors to 90 min of cycle ergometry

Adrenaline infusion of 0.1 g · kg^–1 · min^–1 in healthy volunteers results in an increase of hepatic glucose production, an increase of the absolute number of occupied -adrenoceptors and specific changes in metabolism. To compare these effects with the changes induced by an endogenous catecholamine release, we investigated healthy volunteers during cycle ergometry. After fasting at least 14 h seven healthy subjects exercised for 90 min at an intensity of 20% below their individual anaerobic threshold. The rate of glucose production as well as the turnover rates of alanine and leucine were calculated using stable isotope tracers. High and low affinity -adrenergic binding sites on lymphocytes were determined by an equilibrium binding assay with (–)¹²⁵ Iodocyanopindolol. After 90 min of cycling the rate of appearance of glucose increased significantly from means of 2.0 (SD 0.2) to 2.65 (SD 0.50) mg · kg^–1 · min^–1 with unchanged blood concentrations of glucose and lactate. The flux of the amino acids alanine and leucine decreased significantly from means of 0.91 (SD 0.21) to 0.62 (SD 0.14) mg · kg^–1 · min^–1 and from 0.40 (SD 0.05) to 0.32(SD 0.04) mg · kg^–1 · min^–1, respectively. The mean free fatty acid concentration increased significantly from 0.65 (SD 0.33) to 1.27 (SD 0.45) mmol · l^–1 during the endurance trial. The increase of glucose turnover and the decrease of amino acid flux point to a metabolic shift towards enhanced utilization of free fatty acids. Adrenaline and noradrenaline concentrations showed a moderate but significant increase from means of 0.61 (SD 0.20) to 0.99 (SD 0.36) nmol · l^–1 and from 2.27 (SD 0.75) to 3.46 (SD 0.38) nmol · 1^–1, respectively. The number of high affinity -adrenergic binding sites per cell (-adrenoceptors) nearly doubled from 770 (SD 130) to 1490 (SD 150) during 90 min of cycling. The observed endogenous plasma catecholamine concentrations were not sufficient to change significantly the relative receptor occupancy. This would seem to indicate that the aerobic exercise induced effects depended more on the absolute number of occupied -adrenoceptors than on their relative receptor occupancy. When compared to the results of the adrenaline infusion experiment the increases of the hepatic glucose production and the increase of -adrenoceptors were very similar in both groups despite ten times higher adrenaline plasma concentrations in the infusion group. This would seem to indicate that -adrenoceptors mediated effects do not correlate with catecholamine plasma concentrations.     

Variability of house-dust-mite allergen levels within carpets

Sensitization and exposure to house-dust-mite allergens is an important cause of asthma. Standardized, reliable, and reproducible methods for measuring exposure are essential for the assessment of the relationship between exposure, sensitization, and asthma. This study investigated the variability of the house-dust-mite allergen Der p 1 concentration in reservoir dust collected within whole carpets in living rooms and bedrooms. The carpets of nine bedrooms and 11 living rooms were sampled. Each room was divided into 1 m² areas measured from wall to wall where the carpet was accessible. Reservoir dust samples were collected by vacuuming each 1 m² area for 2 min. Der p 1 was assayed by a two-site monoclonal-antibody-based immunometric ELISA. Der p 1 was detectable in the carpets of nine bedrooms and six of the 11 living rooms. Within these 15 rooms, there was a wide range of Der p 1 levels. The smallest range of allergen within single room was 0.9 μg Der p 1/g dust (0.2 and 1.1 ng/g; 5.5-fold difference), and the largest was 149.2 μg Der p 1/g dust (0.8 and 150 μg/g; 192-fold difference). The mean range of allergen levels in the living rooms was 11.5 jig Der p 1/g of dust, and the mean coefficient of variation of these rooms was 80.2%. illustrating the huge variation of mite allergen levels within each room. The variation within bedrooms was also large, with a mean coefficient of variation value of 88.7%. The coefficient of variation was significantly lower around soft furnishings or beds (57%) than in the rest the room (89.3%), with the mean difference being 32% (95% CI 27ndash;63%; P=0.04). In conclusion, this study has shown that there is a great variation of Der p 1 levels between areas within a room. No consistent pattern of distribution of mite allergen within a room was found. Der p 1 levels in areas around soft furnishings and beds varied less than the levels in the rest of room.     

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into ‘neighborhoods’ comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.     

Familial partial trisomy 5p resulting from segregation of an insertional translocation

A case of duplication of segment p13-p15 of the short arm of chromosome 5 as the result of an insertional translocation in a mentally retarded girl with congenital anomalies is reported. Some of the apparently balanced carriers of the inverted insertion showed minor congenital anomalies.